We aim to develop heart failure (HF) therapies that increase Ca uptake from the myocyte cytoplasm into the sarcoplasmic reticulum (SR). A key abnormality in both experimental and human HF is a defect in SR function, associated with decreased expression and/or specific activity of the SR Ca-ATPase (SERCA2a), the membrane enzyme that pumps Ca2+ into the SR lumen to relax muscle (diastole). Our hypothesis, validated by recent clinical trials, is that activation of SERCA is a powerful approach to treat HF We will increase SERCA activity by direct activation or by relieving inhibition of SERCA by phospholamban (PLB). Over the past decade, the group at Mount Sinai has systematically targeted Ca uptake for heart failure therapy by somatic gene transfer. In animal models of HF, they showed that increasing SERCA2a expression (using rAAV-based gene transfer) improves Ca cycling and contractility. They extended this approach to humans, now in Phase 2b/3 clinical trials, with very promising results for treatment of a wide range of HF syndromes. Thus SERCA activation has been clearly validated for treatment of HF in humans. In parallel, the group at Minnesota has played a leading role in the development of spectroscopic probes for structure- function analysis of SERCA and SERCA-PLB, leading to new mechanistic insights, and to the engineering of fluorescent biosensors that provide direct detection of functionally important SERCA structural changes and PLB interactions in living cells. In collaboration, the MN and MS groups have shown that these biosensors, used with novel fluorescence lifetime instrumentation, are powerful tools for designing small molecules or PLB mutants (PLBM) with potential for therapeutic activation of SERCA. In this project, the MN and MS groups will intensify their collaboration to activate SERCA for HF therapy. Two complementary approaches will be used. In Aim 1, we seek small molecules that activate SERCA directly (1a) or disrupt the inhibitory SERCA-PLB interaction (1b). In Aim 2, we pursue a gene therapy approach, using structure-based design to engineer PLB mutants that bind to SERCA with high affinity but do not inhibit the enzyme, thus displacing endogenous PLB and relieving SERCA inhibition. These projects take advantage of complementary world-class expertise in the two research teams - the Minnesota team will provide the structure-based insights and fluorescence-based screening methods to identify therapeutic candidates, and the Mount Sinai Group will test these reagents in animal models of HF. Based on excellent preliminary data and the unique capabilities of these two teams, expected outcomes are small molecules (lead compounds) and PLB mutants ready for Phase I clinical trials within 5 years.